This application is in response to PA-03-080 as an exploratory/developmental R21 grant. Herein, we propose to develop a new synthetic saponin adjuvant to be used with the Marburg glycoprotein (MBGV GP) as a vaccine preparation. Since Marburg virus (MBGV) is one of the most deadly viruses that can be used as a bioterrorism agent (i.e., a NIAID Category A priority pathogen), there is an urgent need to develop a potent vaccine for this agent. Adjuvants not only play a crucial role as delivery vehicles, but they also stimulate humoral and cell-mediated immunity characterized by cytolytic T-lymphocytes, which is critical for the generation of an effective immune response and long term protective immunity against viral infections. Saponins, especially from Gypsophilla species, Saponaria officinalis, and Quillaja saponaria Molina, possess potent immunomodulating activity (both humoral and cell mediated) in humans for bacterial, viral, and other infections. However, their use is limited due to recognized toxicity and stability issues. We propose the synthesis of novel saponins from gypsogenic acid (aglycone) utilizing the established structure-activity data of natural saponins, QS-21, and a semi-synthetic saponin preparation, GPI-0100. These new adjuvants will be screened for their efficacy in a mouse model using ovalbumin (OVA). The most potent new adjuvant will then be evaluated for its stability and acute toxicity. It will also be evaluated in a guinea pig model using OVA. The data thus obtained will be provided to Dr. Hevey at USAMRIID. He will utilize the best adjuvant in a MBGV GP vaccine preparation and evaluate it in a guinea pig model using lethal aerosol challenge with MBGV, followed by lethal challenge of survivors by the subcutaneous route. The development of a well-characterized single saponin species will not only provide a potentially useful new vaccine for Marburg, but should have application for important vaccines against other infectious agents. In addition, these studies will provide direction for the second-generation development of improved saponin adjuvants. Furthermore, their use as fluorescent probes should help elucidate the mechanism of action of this class of adjuvants, increasing our understanding of how to better modulate an immune response and better protect against dangerous pathogens.